Existence of transient functional double-stranded DNA intermediates during recombinant AAV transduction

Abstract

Previous studies have documented that 0.1Ϸ1% of input recombinant adeno-associated virus (rAAV) vectors could be stabilized and lead to transgene expression. To characterize the steps involving massive AAV DNA loss, we designed an''AAV footprinting'' strategy that can track newly formed AAV dsDNA genomes. This strategy is based on an ROSA26R mouse model or cell line that carries a lacZ gene flanked by two loxP sites. When it is transduced by a rAAV vector carrying the Cre recombinase, the lacZ gene can be activated and remain active even when rAAV genomes are later lost. By using this sensitive AAV footprinting technique, we confirmed the existence of transient AAV dsDNA that went undetected by conventional DNA methods. Although these dsDNA intermediates could be efficiently formed in almost every cell and were competent for mRNA transcription and protein synthesis in vivo, they got lost continuously. Only a small fraction was eventually stabilized for sustained gene expression. Although both rAAV2 and rAAV8 can potentially have similar levels of dsDNA formation, AAV8 dsDNA was formed much faster than that of AAV2, which explains why rAAV8 is more efficient than rAAV2 in transducing the liver. Collectively, our studies suggested that rather than receptor binding, viral entry, and ssDNA to dsDNA conversion, the instability of newly formed AAV dsDNA was the primary contributing factor for the low rAAV transduction efficacy. The uncoating step significantly influenced the stability of AAV transient dsDNA. The identification of transient AAV dsDNA provided a new pathway for improving rAAV transduction. adeno-associated virus ͉ DNA stability ͉ gene therapy ͉ vector ͉ ROSA26R

Native Molecular State of Adeno-Associated Viral Vectors Revealed by Single-Molecule Sequencing

“…Recombinant AAV vectors were produced by the tripletransfection method, which has been described previously (Wang et al, 2007). Briefly, a vector plasmid (with AAV2 inverted terminal repeats), a helper plasmid (with the AAV2 rep and cap genes), and mini-adenovirus helper plasmid (pFD6, with essential regions from the adenovirus genome) were cotransfected into 293 cells by calcium phosphate precipitation.…”

Mutagenesis of Adeno-Associated Virus Type 2 Capsid Protein VP1 Uncovers New Roles for Basic Amino Acids in Trafficking and Cell-Specific Transduction

“…The vast majority of genomes remain outside the nuclear membrane (12,29), but ultimately, the transgene must be delivered to the nucleus, transition from a single-stranded genome to a double-stranded intermediate, and convert to a stable double-stranded form before transduction is considered complete (62). We have previously shown that capsids are capable of entering the nucleus intact (33), but the precise location for uncoating is still debated.…”

Recombinant AAV2-mediated β-globin expression in human fetal hematopoietic cells from the aborted fetuses with β-thalassemia major

“…These low levels of b-chain expression may be attributed to a relative short period (up to 70 days), because AAV2-mediated gene expression usually relies on slow and steady formation of double-stranded DNA by strand annealing [31,32]. By extending the period of observation, genetically modified hematopoietic cells may increase expression of b-globin by the gradual conversion of the input singlestranded DNA to transcriptionally active double-stranded forms.…”